McCormack J G, Bromidge E S, Dawes N J
Department of Biochemistry, University of Leeds, U.K.
Biochim Biophys Acta. 1988 Jul 27;934(3):282-92. doi: 10.1016/0005-2728(88)90088-6.
The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat kidney mitochondria were found to be essentially similar to those described previously for other mammalian tissues; in particular each enzyme could be activated severalfold by Ca2+ with half-maximal effects (K0.5 values) of about 1 microM and effective ranges of approx. 0.1-10 microM Ca2+. In intact mitochondria prepared from whole rat kidneys incubated in a KCl-based medium containing respiratory substrates, the amount of active, nonphosphorylated pyruvate dehydrogenase could be increased severalfold by increases in extramitochondrial [Ca2+]; these effects could be blocked by ruthenium red. Similarly, Ca2+-dependent activations of NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase could be demonstrated in intact, fully coupled, rat kidney mitochondria by either following O2 uptake (in the presence of ADP) and NAD(P)H reduction (in the absence of ADP) on presentation of non-saturating concentrations of either threo-Ds-isocitrate or 2-oxoglutarate, respectively, under appropriate conditions, or for the latter enzyme only, also by following 14CO2 production from 2-oxo[1-14C]glutarate (in the absence or presence of ADP). Effects of Na+ (as a promoter of egress) and Mg2+ (as an inhibitor of uptake) on Ca2+-transport by rat kidney mitochondria could be readily demonstrated by assaying for the Ca2+-sensitive properties of the intramitochondrial Ca2+-sensitive dehydrogenases within intact rat kidney mitochondria. In the presence of physiological concentrations of Na+ (10 mM) and Mg2+ (2 mM), activation of the enzymes was achieved by increases in extramitochondrial [Ca2+] within the expected physiological range (0.05-5 microM) and with apparent K0.5 values in the approximate range of 300-500 nM. The implications of these results on the role of the Ca2+-transport system of kidney mitochondria are discussed.
研究发现，大鼠肾脏线粒体提取物中钙敏感的线粒体内酶（丙酮酸脱氢酶磷酸酶、NAD⁺-异柠檬酸脱氢酶和2-氧代戊二酸脱氢酶）的调节特性与先前描述的其他哺乳动物组织基本相似；特别是每种酶都可被Ca²⁺激活数倍，半数最大效应（K0.5值）约为1微摩尔，有效范围约为0.1 - 10微摩尔Ca²⁺。在用含呼吸底物的KCl基培养基孵育的全大鼠肾脏制备的完整线粒体中，线粒体外[Ca²⁺]的增加可使活性、非磷酸化的丙酮酸脱氢酶量增加数倍；这些效应可被钌红阻断。同样，在完整、完全偶联的大鼠肾脏线粒体中，通过在适当条件下分别加入非饱和浓度的苏糖-Ds-异柠檬酸或2-氧代戊二酸后跟踪O₂摄取（存在ADP时）和NAD(P)H还原（不存在ADP时），或者仅针对后一种酶，也通过跟踪2-氧代[1-¹⁴C]戊二酸产生的¹⁴CO₂（不存在或存在ADP时），可以证明NAD⁺-异柠檬酸脱氢酶和2-氧代戊二酸脱氢酶的Ca²⁺依赖性激活。通过测定完整大鼠肾脏线粒体内线粒体内钙敏感脱氢酶的钙敏感特性，可以很容易地证明Na⁺（作为流出促进剂）和Mg²⁺（作为摄取抑制剂）对大鼠肾脏线粒体钙转运的影响。在生理浓度的Na⁺（10 mM）和Mg²⁺（2 mM）存在下，通过线粒体外[Ca²⁺]在预期生理范围内（0.05 - 5微摩尔）增加并使表观K0.5值在约300 - 500 nM范围内，实现了酶的激活。讨论了这些结果对肾脏线粒体钙转运系统作用的影响。
